WO2004033626A2 - Hcg hyperglycosyle (antigene des trophoblastes invasifs) utilise dans le diagnostic differentiel de la maladie trophoblastique maline ou invasive - Google Patents

Hcg hyperglycosyle (antigene des trophoblastes invasifs) utilise dans le diagnostic differentiel de la maladie trophoblastique maline ou invasive Download PDF

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WO2004033626A2
WO2004033626A2 PCT/US2003/021306 US0321306W WO2004033626A2 WO 2004033626 A2 WO2004033626 A2 WO 2004033626A2 US 0321306 W US0321306 W US 0321306W WO 2004033626 A2 WO2004033626 A2 WO 2004033626A2
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hcg
biological sample
ita
cell tumor
germ cell
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WO2004033626A3 (fr
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Laurence A. Cole
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Science & Technology Corporation @ Unm
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57442—Specifically defined cancers of the uterus and endometrial
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Definitions

  • the invention relates to methods and reagents for differential diagnosis of quiescent and invasive gestational trophoblastic diseases.
  • the invention also relates to methods for detecting the presence or absence of germ cell tumors.
  • a gestational trophoblastic disease is characterized by abnormal cellular growth of the tissues formed as a result of conception, leading to the development of tumors within the uterus.
  • PSTT placenta-site trophoblastic tumor
  • a gestational trophoblastic disease is diagnosed by ultrasound or a Computerized Axial Tomography (CT) or Magnetic Resonance Imaging (MSI) imaging methods.
  • CT Computerized Axial Tomography
  • MSI Magnetic Resonance Imaging
  • hCG human chorionic gonadotropin
  • hCG levels are often rechecked multiple times if a trophoblastic tumor is suspected, since they are a direct measure of the amount of tumor tissue present.
  • treatment for gestational trophoblastic disease includes chemotherapy, surgery and combination chemotherapy, or a combination thereof.
  • the treatment or combination of treatments that is recommended will depend upon the severity of the disease.
  • a gestational trophoblastic disease can be invasive, which will require a more rigorous treatment regimen, or non-invasive, which will not require any of the treatments mentioned above.
  • non-invasive or slow growing none of the above mentioned treatments would be effective since they act on fast growing tumors.
  • there is currently no accurate method for distinguishing between invasive and non-invasive gestational trophoblastic disease there is a need in the art for a method of diagnosing non-invasive gestational trophoblastic disease in order to spare a patient from undergoing unnecessary treatments used to treat invasive gestational trophoblastic disease.
  • the invention provides methods for detecting the presence or absence of invasive trophoblast cells comprising the steps of: (a) obtaining a biological sample from a patient; (b) measuring an amount of hCG in the biological sample; (c) measuring an amount of IT A in the biological sample; and (d) determining the percentage of hCG that is IT A, wherein invasive trophoblast cells are detected if the percentage of hCG that is ITA is 30% or greater.
  • the patient is a patient in which persistently low hCG titers have been previously detected, and wherein quiescent gestational trophoblastic disease is diagnosed if the percentage of ITA relative to hCG determined in step (d) is less than 30%.
  • the invention provides methods for detecting the presence or absence of invasive trophoblast cells in a biological sample comprising the steps of: (a) obtaining a biological sample from a patient; and (b) measuring an amount of ITA in the biological sample; wherein invasive trophoblast cells are detected if the amount of ITA in the biological sample is 2 IU/L or greater.
  • the invention provides methods for monitoring progression of quiescent gestational trophoblastic disease comprising the steps of: (a) obtaining a biological sample from a patient diagnosed as having quiescent gestational trophoblastic disease; (b) measuring an amount of hCG in the biological sample; (c) repeating steps (a) and (b) with a biological sample obtained at one or more subsequent time points; (d) measuring an amount of ITA in a biological sample obtained in step (c) where the amount of hCG in the biological sample firom step (c) is higher than the amount of hCG in step (b); and (e) determining a percentage of hCG that is ITA in the biological sample from step (d).
  • the invention provides methods of detecting the presence or absence of a germ cell tumor in a biological sample comprising the steps of: (a) obtaining a biological sample from a patient; (b) measuring an amount of hCG in the biological sample; (c) measuring an amount of ITA in the biological sample; and (d) determining the percentage of hCG that is ITA, wherein a germ cell tumor is detected if the percentage is 30% or greater.
  • the invention provides methods of detecting the presence or absence of a germ cell tumor in a biological sample comprising the steps of: (a) obtaining a biological sample from a patient; and (b) measuring an amount of ITA in the biological sample; wherein a germ cell tumor is detected if the amount of ITA in the biological sample is 2 IU/L or greater.
  • the invention provides methods of monitoring the progression of a germ cell tumor comprising the steps of: (a) obtaining a biological sample from a patient diagnosed as having a germ cell tumor; (b) measuring an amount of hCG in the biological sample; (c) repeating steps (a) and (b) with a biological sample obtained at subsequent time points; (d) measuring an amount of ITA in a biological sample from step (c) if the amount of hCG in a biological sample from step (c) is higher than the amount of hCG in step (b); and (e) determining the percentage of hCG that is ITA in the biological sample from step (d).
  • a germ cell tumor detected or monitored by a method of the invention can be an ovarian germ cell tumor or a testicular germ cell tumor.
  • Figure 1 is a chart on use of invasive trophoblastic antigen (ITA) as a tumor marker in selected patient populations.
  • ITA invasive trophoblastic antigen
  • Table 1 represents the percentages of ITA in 38 patients with persistent low levels of hCG as described in Table 1; (2) represents the percentages of ITA in 4 patients re-referred to the USA hCG Reference Service, all of whom initially presented with persistent low levels of hCG and were either shown or clinically suspected of having malignant gestational trophoblastic disease when sudden rapidly rising hCG levels were detected; and (3) represents 15 other cases with proven gestational trophoblastic disease (choriocarcinoma or invasive placental site trophoblastic tumor).
  • the value 100% indicates that the ITA assay was >99% of that in the total hCG assay (which measures all forms of hCG including ITA).
  • ITA units are equal on a mass basis to hCG units (IU/L).
  • hCG is an abbreviation for human chorionic gonadotropin, a glycoprotein hormone secreted in relatively large quantities by the trophoblast cells of the placenta.
  • intact or "regular" hCG is used herein to refer to hCG that is composed of two dissimilar subunits, c. (92 amino acids and two N-linked oligosaccharides) and ⁇ (145 amino acids and two N-linked and four O-linked oligosaccharides), that are joined noncovalently. Both subunits are extensively glycosylated leading to a heterogeneous population of molecular forms.
  • hCG can be detected in the serum and urine of pregnant women and in those with gestational trophoblastic disease. Free - and free ⁇ -subunits are also detected in serum and urine samples. Both subunits undergo extensive glycosylation, creating a mixture of various forms of hCG and free hCG subunits in a sample. Furthermore, free ⁇ subunit and ⁇ subunits of intact hCG are frequently cleaved at position 47-48 to produce nicked hCG and nicked ⁇ subunit, which are biologically inactive.
  • ITA is an abbreviation for invasive trophoblast antigen, also known as hyperglycosylated hCG.
  • ITA is, therefore, a variant of regular hCG, comprising additional side chains of sugars on the N-linked and O-linked sugar chains compared with regular hCG. These additional sugar side chains comprise N- acetylglucosamine, galactose and sialic acid and make the molecule considerably larger in size.
  • ITA is best detected with an immunoassay using the specific monoclonal antibody B152, which only detects ITA.
  • ITA is produced by invasive cytotrophoblast cells and malignant germ cells.
  • measuring ITA levels in a patient who is not pregnant and/or is free of a disease that has cells producing ITA will yield a background level of ITA of less than 2 IU/L.
  • an ITA level of 2 IU/L or greater is indicative of a cancer.
  • a level of ITA that is 2 IU/L or greater in a biological sample can indicate the presence of invasive trophoblast cells and/or a germ cell tumor, wherein a level of ITA that is lower than 2 IU/L can indicate the absence of invasive trophoblast cells and/or a germ cell tumor.
  • hCG is used herein to refer to any subunit of hCG, fragment of hCG, intact hCG, an isoform of hCG, a modified hCG molecule, total hCG, or any combination thereof.
  • a subunit of hCG can be, for example, or ⁇ subunits.
  • a fragment of hCG can be, for example, ⁇ -core fragment or hCG missing the ⁇ -subunit C-terminal peptide.
  • a modified hCG molecule can be, for example, nicked free ⁇ -subunit or nicked intact hCG.
  • An isoform of hCG can be, for example, ITA.
  • total hCG comprises regular hCG and all forms of hCG, including free c. and ⁇ subunits of hCG and ⁇ core fragment of hCG, as well as hCG isoforms such as ITA.
  • ITA hCG isoforms
  • quiescent gestational trophoblastic disease is used herein to describe a dormant syndrome characterized by non-invasive or quiescent trophoblastic tissue, failed response to chemotherapy and surgery, and the retention of some malignant potential.
  • the term "gestational trophoblastic disease,” as used herein, includes hydatidifonn mole/molar pregnancy, choriocarcinoma, and placenta-site trophoblastic tumor.
  • Germ cell tumor refers to a tumor arising from ovarian germ cells or testicular germ cells.
  • Germ cell tumors that produce ITA include, for example, dysgerminoma and other ovarian germ cell tumors, and testicular choriocarcinoma and seminoma.
  • ITA is a useful marker in detecting invasive disease in all these genn cell tumors or malignancies.
  • biological sample includes, but is not limited to, a quantity of a tissue or body fluid sufficient to measure hCG and/or ITA levels, that is obtained from a mammal.
  • the mammal is a human.
  • a biological sample is urine, saliva, cells, plasma, or serum. Most preferably, a biological sample is urine.
  • the term "patient” includes mammalian subjects.
  • a patient is a human.
  • the patient is a male or female in which persistently low hCG titers have been previously detected, a female diagnosed as having quiescent gestational trophoblastic disease, a female at risk of having or suspected of having a gestational trophoblastic disease, or a male or female at risk of having or suspected of having or in need of screening for a germ cell tumor.
  • a "low level" of hCG is detected where the concentration of hCG is 2 to 200 IU/L.
  • a "persistently" low level of hCG is detected where a low level of hCG is detected in biological samples obtained from a single patient at subsequent time intervals, for example, at weekly, monthly, or bi-monthly time intervals.
  • the term "detecting” refers to identifying a patient with invasive trophoblast cells or a germ cell tumor and/or a patient who is at high risk for having an invasive gestational trophoblastic disease or a germ cell tumor.
  • the invention provides a method of detecting the presence or absence of invasive trophoblast cells comprising the steps of: (a) obtaining a biological sample from a patient; (b) measuring an amount of hCG in the biological sample; (c) measuring an amount of ITA in the biological sample; and (d) determining a percentage of hCG that is ITA, wherein invasive trophoblast cells are detected if the percentage of hCG that is ITA is at least 30%.
  • the percentage of hCG that is ITA can be at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • invasive trophoblast cells are detected if the percentage of hCG that is ITA is at least 30% or greater.
  • hCG can be regular hCG, a subunit of hCG, an isoform of hCG, a fragment of hCG, a combination thereof, or total hCG.
  • Methods for measuring an amount of hCG in a sample are known in the art, and include, for example, using commercially available hCG tests. Such tests have been described, for example, in Cole, 1998, Gynecol. Oncol. 71:325-329; Rotmensch et al, 2000, Lancet 355:712-715; Cole et al, 2001, Clin. Chem. 47:308-315; Cole et al., 2001, Clin. Lab. Intl. 25:9-14; Cole et al., 2002, J Reprod. Med. 47:433-444; Butler et al, 2001, Clin. Chem. 47:1332-1333; Butler et al, 2002, Obstet. Gynecol.
  • Methods for measuring an amount of ITA include, for example, methods described in U.S. Patent No. 6,429,018 (which is hereby incorporated by reference), ITA kits that are available, for example, from Quest Diagnostics (Teterboro, NJ) and Nichols Institute Diagnostics (San Clemente, CA), and methods described herein.
  • Any assay that functions to qualitatively or quantitatively determine concentrations of hCG in a biological sample and/or detects ITA concentrations can be employed in the practice of the invention.
  • a direct assay such as an immunoassay using antibodies that recognize hCG, free hCG subunits, the hCH ⁇ -core fragment, and/or specific hCG isoforms, is preferred.
  • exemplary assays can involve lectins that assay for carbohydrate moieties, chromatography, chemical or electrophoresis or isoelectric focusing tests that detect glycosylation variants of hCG, and/or antibodies to hyperglycosylated or carbohydrate-variant hCG. Such assays are described in the art.
  • Irnmunoassays that can be used to detect hCG or ITA include, but are not limited to, assays employing specific antibodies to hCG or ITA, and assays employing nonspecifically defined antibodies obtained by blind injections of a hCG or ITA into test animals using standard methods.
  • Antibodies to hCG and ITA can be generated by standard means as described, for example, in "Antibodies: A Laboratory Manual” by Harlow and Lane (Cold Spring Harbor Press, 1988), which is hereby incorporated by reference. Any type of fusion phage, monoclonal, or polyclonal antibodies can be used in irnmunoassays of the invention, so long as the antibodies can be used in a reproducible fashion as markers for hCG or ITA.
  • an amount of hCG or ITA can be measured using a capture antibody followed by a labeled secondary antibody using a strategy as described, for example, in U.S. Patent No. 6,429,018 (which is hereby incorporated by reference).
  • U.S. Patent No. 6,429,018 teaches the B152 antibody that recognizes ITA but does not detect normal hCG.
  • a labeled secondary antibody useful in a method of the invention can be, for example, an anti-hCG antibody, ⁇ -core fragment, ⁇ -subunit, and/or ⁇ -subunit, providing an assay with polypeptide specificity.
  • the label on the secondary antibody can comprise any chemical, radioactive, lanthanide, colored dye, or genetic tag used in enzyme-linked immunosorbent assays (ELISAs), Western blots, and other sensitive and specific irnmunoassays and immunoradiometric assays using known methodology. These include conjugating the antibody with horseradish peroxidase or alkaline phosphatase that are easily measurable, typically using colorimetric, fluorometric or luminescent substrates. Genetic labels include firefly luciferase, employed because luciferase produces a biol uminescent molecule when incubated with its substrate, luciferin.
  • hCG peptide-specific antibody can be used as a capture antibody, and an antibody specific to ITA can be used as the secondary labeled antibody in an immunoassay such as those described above.
  • Competitive irnmunoassays can also be used to determine the amount of ITA present among other forms of hCG, including regular and total hCG.
  • an antibody that binds non-specifically to both intact hCG and ITA can be used to immunoprecipitate both forms of hCG present in a sample, followed by a second immunoprecipitation step in which ITA-specific antibodies are used to separate ITA from regular hCG, free hCG subunits, and hCG ⁇ core fragment.
  • Isolated ITA protein can then be quantified using standard methods, such as a Bradford protein assay. Alternate embodiments using concanavalin A or other carbohydrate-specific lectin can be used in place of the capture antibody or labeled antibody. Alternatively, prior to an immunoassay, a lectin or chromatographic method can be used to extract ITA from a biological sample. Lectins bind specific carbohydrate structures. Thus, they can be used to differentiate ITA and other forms of hCG, including regular hCG and various hCG subunits or hCG fragments. ITA is also slightly larger than regular hCG and can be separated from regular hCG or other hCG forms by chromatography or elec trophoresis.
  • Carbohydrate analyses include qualitative observations of differences in physical properties between regular hCG and ITA or subunits and fragments thereof, carbohydrate identification using plant lectins specific to the variant carbohydrate portion of ITA obtained by standard lectin screening methods, or any other fingerprinting technique including qualitative or quantitative carbohydrate composition analyses. See, for example, U.S. Patent No. 6,429,018, which is incorporated by reference.
  • hCG can be purified from biological samples prior to determining the percentage of hCG that is ITA from the sample. Any method for purifying hCG can be used. For example, antibodies specific for hCG can be used to isolate hCG from a biological sample. The amount of ITA in a purified hCG protein fraction can be determined using a method as described above, for example, using an immunoassay with antibodies specific for ITA. ITA specific antibodies are described, for example, in U.S. Patent No. 6,429,018, which is hereby incorporated by reference. Purified hCG and hCG isoforms from control or test samples can be stored under appropriate conditions, such as those described, for example, in Cole et al, 1999, Clinical Chem. 45:2109-2119, which is hereby incorporated by reference.
  • the invention provides a method of diagnosing quiescent gestational trophoblastic disease or invasive gestational trophoblastic disease in a patient comprising the steps of: (a) obtaining a biological sample from a patient, wherein the patient has persistently low hCG titers; (b) measuring an amount of hCG in the biological sample; (c) measuring an amount of ITA in the biological sample; and (d) determining the percentage of hCG that is ITA, wherein quiescent gestational trophoblastic disease is diagnosed if the percentage determined in step (d) is less than 30%, and invasive gestational trophoblastic disease is diagnosed if the percentage determined in step (d) is greater than 30%.
  • hCG can be regular hCG, a subunit of hCG, an isoform of hCG, a fragment of hCG, a combination thereof, or total hCG.
  • the amount of ITA from step (c) can be compared to a predetermined value, wherein the patient is diagnosed as having invasive gestational trophoblastic disease if the amount of ITA is equal to or greater than the predetermined value, and wherein the patient is diagnosed as having quiescent gestational trophoblastic disease if the amount of ITA is less than the predetermined value.
  • the predetermined value is 2 IU/L, as discussed above. Certain embodiments of this aspect are as defined above, including the definition of patient and persistently low levels of hCG, biological sample, quiescent trophoblastic disease, ITA, and hCG.
  • a predetermined value is a standardized value based on a control.
  • a predetermined value can be based on an amount of ITA that is present in a biological sample obtained from a woman previously diagnosed with quiescent gestational trophoblastic disease, i.e. a woman having persistently low hCG levels without a sharp rise in hCG levels.
  • a "persistently low hCG levels” are defined above.
  • a “sharp rise" in hCG levels occurs when an hCG level observed in a biological sample is at least 2 fold greater than an hCG level observed in a biological sample obtained from the same patient at an earlier time point. For example, a sharp rise is observed where hCG levels increase from 5 IU/ml in one biological sample to 10 IU/ml in a subsequently obtained biological sample, wherein the two biological samples were obtained from the same patient.
  • the "predetermined value" can refer to a value that is based on an amount of ITA in a biological sample obtained from a woman who has confirmed invasive gestational trophoblastic disease, i.e. a woman who has been pathologically diagnosed as having choriocarcinoma.
  • the patient is diagnosed as having invasive gestational trophoblastic disease if the amount of ITA is equal to or greater than the predetermined value.
  • the invention provides methods for monitoring the progression of quiescent gestational trophoblastic disease comprising the steps of: (a) obtaining a biological sample from a patient; (b) measuring an amount of hCG in the biological sample; (c) repeating steps (a) and (b) with a biological sample obtained at subsequent time points; (d) measuring an amount of ITA in the biological sample if the amount of hCG in the biological sample from step (c) is higher than the amount of hCG in step (b); and (e) determining the percentage of hCG that is ITA in the biological sample.
  • the patient can be a patient who was previously diagnosed as having quiescent gestational trophoblastic disease, as diagnosed, for example, using a method of the invention.
  • the patient can be a patient who was previously treated for an invasive gestational trophoblastic disease and whose hCG levels subsequently decreased relative to hCG levels measured prior to treatment for invasive gestational trophoblastic disease.
  • Certain embodiments of this aspect are as defined above, including the definition of patient and persistently low levels of hCG, biological sample, quiescent trophoblastic disease, ITA, and hCG.
  • steps (a) and (b) can be repeated, for example, daily, weekly, monthly, bi-monthly, every other month, every three months, every four months, every six months, or yearly.
  • quiescent disease is indicated where hCG levels observed over time rise then plateau.
  • quiescent disease is indicated where hCG levels rise to 20 IU/ml, then plateau at 22, 18, 25, and 24 IU/ml.
  • invasive disease is indicated where hCG levels rise consistently or sharply.
  • invasive disease is indicated where hCG levels rise to 20 IU/ml, then continue to rise to 100 IU/ml then 600 IU/ml.
  • invasive disease is confirmed in a patient having consistently or sharply rising hCG levels by determining the percentage of ITA relative to the total amount of hCG present in a biological sample from the patient. Where the amount of ITA is at least 30% or higher, invasive disease is diagnosed.
  • hCG can be regular hCG, a subunit of hCG, an isoform of hCG, a fragment of hCG, a combination thereof, or total hCG.
  • the methods of the invention can be used to diagnose and/or monitor progression of quiescent gestational trophoblastic disease or invasive gestational trophoblastic disease.
  • One recommended management of quiescent disease is close surveillance without therapy until invasive disease is detected.
  • the invention provides the following guidelines for the management and monitoring of quiescent gestational trophoblastic disease.
  • a patient having persistent low levels of serum hCG and/or an ITA amount that is less than 30% of the patient's hCG is given the diagnosis of quiescent gestational trophoblastic disease instead of invasive or malignant gestational trophoblastic disease .
  • a patient with quiescent gestational trophoblastic disease should be monitored for an abrupt rise in hCG titers, ITA titers, and/or ITA to hCG ratios, which could occur months or years after the detection of persistent low-level hCG titers.
  • the invention provides methods of detecting the presence or absence of a germ cell tumor in a biological sample comprising the steps of: (a) obtaining a biological sample from a patient; (b) measuring an amount of hCG in the biological sample; (c) measuring an amount of ITA in the biological sample; and (d) determining the percentage of hCG that is ITA, wherein a germ cell tumor is detected if the percentage is 30% or greater.
  • hCG can be regular hCG, a subunit of hCG, an isoform of hCG, a fragment of hCG, a combination thereof, or total hCG.
  • the invention provides methods of detecting the presence or absence of a germ cell tumor in a biological sample comprising the steps of: (a) obtaining a biological sample from a patient; and (b) measuring an amount of ITA in the biological sample; wherein a germ cell tumor is detected if the amount of ITA in the biological sample is 2 IU/L or greater.
  • the invention provides methods of monitoring the progression of a genn cell tumor comprising the steps of: (a) obtaining a biological sample from a patient diagnosed as having a germ cell tumor; (b) measuring an amount of hCG in the biological sample; (c) repeating steps (a) and (b) with a biological sample obtained at subsequent time points; (d) measuring an amount of ITA in a biological sample from step (c) if the amount of hCG in a biological sample from step (c) is higher than the amount of hCG in step (b); and (e) determining the percentage of hCG that is ITA in the biological sample from step (d).
  • hCG can be regular hCG, a subunit of hCG, an isoform of hCG, a fragment of hCG, a combination thereof, or total hCG.
  • Samples received in a reference laboratory were initially tested using the automated DPC Immulite total hCG test, the DPC Immulite free hCG ⁇ test, Abbott AxSym total hCG test, and other commercial hCG tests as needed. Serum and urine samples were also analyzed using specialized assays. These included the microtiter plate intact hCG only enzyme immunometric assay, the ITA enzyme immunometric assay, the nicked hCG only enzyme immunometric assay, the free hCG ⁇ -subunit only enzyme immunometric assay, and the hCG ⁇ -subunit core fragment only enzyme immunometric assay. These specialized tests have been described in detail in previous publications (1- 8). Tests were run with and without the Scantibodies, Inc.
  • Table 1 outlines the clinical histories provided for the 63 patients with persistent low-level hCG determinations.
  • the table shows the antecedent gestation, the period of persistent low serum hCG results, the limits of serum hCG results as determined by the referring physician's laboratory and the reference laboratory, and therapy used for treating the low hCG results, as indicated in reports to the reference laboratory.
  • methotrexate Act-D, actinomycin D; EMA-CO, combination etoposide, metho trexate, actinomycin D alternating with cyclophosphamide, and vincris tine chemotherapy; Taxol, paclitaxel; EIP, combination etoposide, ifosphamide, and cisplatin; EIC; combination etoposide, ifosphamide, and carboplatin; MtxLv, methotrexate with leucovorin rescue; 5FU, 5-flurouracil; Mtx, Mtx, 2 separate treatments with methotrexate; GTN, gestational trophoblastic neoplasm.
  • hCG result was then 355 IU/L.
  • 100% ofhCG immunoreactivity due to ITA Figure 1
  • patient was found to have invasive placental trophoblastic tumor.
  • the clinically recognized pregnancy event before this presentation was a hydatidifonn mole in 27 cases (43%); 35 (56%) had a term, ectopic, or spontaneously aborted pregnancy; one case had a past history of gestational trophoblastic neoplasm.
  • hCG titers In four cases (6.3%), hCG titers abruptly and steeply increased, after prolonged surveillance (after 1, 2, 3, and 4.5 years, respectively) of persistent, low-level hCG titers. In 3 of the 4 cases, the antecedent event was a pregnancy, and in one case was a complete hydatidifonn mole. All 4 patients were again referred to the reference laboratory for further evaluation. At this juncture, real hCG titers substantially higher than previous results were demonstrated (Table 1, cases 7, 14, 18 and 34). In one patient tumor was identified in the right lower lobe of the lung by CT imaging. Metastatic placental site trophoblastic tumor was confirmed by pathologic inspection.
  • This patient was given EMA-CO combination chemotherapy (etoposide, methotrexate and actinomycin C alternating with cyclophosphamide and vincristine) without complication.
  • tumor was imaged in the uterus by magnetic imaging in one case.
  • CT and magnetic resonance imaging did not identify a tumor.
  • invasive gestational trophoblastic neoplasm or choriocarcinoma was clinically suspected.
  • ITA has the sensitivity and specificity to differentiate a pre-invasive form with low hCG titers, or quiescent gestational trophoblastic disease, from malignant or invasive gestational trophoblastic disease.
  • the diagnosis of persistent gestational trophoblastic disease was rendered, although no tumor could be identified by CT or magnetic resonance imaging.
  • Most cases (64%) received single or multi-agent chemotherapy, surgery, or a combination.
  • measurable circulating hCG persisted.
  • no or minimal immunoreactivity was identified. This indicated the absence or minimal presence of invasive cytotro phoblastic cells.
  • hCG Reference Service Forty cases were identified at the USA hCG Reference Service (Albuquerque, New Mexico) with low values of real (not false positive) hCG persisting with small variations in hCG results for period ranging from 4 months to 12 years (Table 3). In all cases no tumor was found by ultrasound, CT or MRI scans. In each case persistent gestational trophoblastic disease had been assumed, and in 25 of the 40 cases (63%) referred to the hCG Reference Service (no information available on 2 of the 40 cases) chemotherapy, combination chemotherapy (EMACO) or hysterectomy had been performed. In all cases the therapy failed to completely suppress the hCG values. In all 40 cases real hCG was detected in serum. Table 3
  • hCG result was 6000 IU/L.
  • 81% of the total hCG immunoreactivity was shown to be due to hyperglycosylated hCG (Table 4).
  • Malignant disease (gestational trophoblastic disease/choriocarcinoma) was identified and chemotherapy commenced.
  • hCG result was 489 IU/L.
  • 100% of the total hCG immunoreactivity was shown to be due to hyperglycosylated hCG (Table 4).
  • Malignant disease (gestational trophoblastic disease/choriocarcinoma) was indicated and chemotherapy commenced.
  • the USA hCG Reference service also consulted on 40 other cases with persistent low hCG values but with history of pregnancy and no history of trophoblastic diseases whatsoever (Table 5). In these cases, persistent low hCG levels were detected from 2 month to 6 years. In all 40 cases hCG was identified in both serum and urine samples. The hCG resembled normal second or third trimester pregnancy hCG, with very little or no ITA ( ⁇ 2 IU/L or ⁇ 30% of total hCG). In all 40 cases no tumor mass was identified. Little or no response was observed to chemotherapy.
  • hCG hypoglycosylated hCG not detected
  • values sharply rose.
  • USA hCG Reference Service was consulted.
  • hCG was 863 IU/L, 86% due to hyperglycosylated hCG (Table 4).
  • Malignancy (gestational trophoblastic disease/choriocarcinoma) was demonstrated.
  • hCG was 1255 IU/L, -100% due to hyperglycosylated hCG (Table 4). Malignancy (gestational trophoblastic disease/choriocarcinoma) was demonstrated.
  • the USA hCG References Service observed 80 cases of persistent low levels of hCG, whether with history of hydatidifonn mole or gestational trophoblastic disease (quiescent gestational trophoblastic disease) or with history of only pregnancy (unexplained elevated hCG).
  • hCG results rapidly rose, indicating active disease.
  • PSTT was detected and in six cases gestational trophoblastic disease/choriocarcinoma was diagnosed and treated. This observation indicated that persistent low levels of hCG may be a pre- malignant condition.
  • ITA results were 57% to -100% of total hCG (Table 4). Putting these cases together, ITA accounted for more than 30% of total hCG immunoreactivity in 20 of 20 malignant cases, and in none of the 53 individuals with persistent elevated hCG (non-invasive disease). The data indicated that the percent ITA is an accurate indicator of invasive/malignant trophoblastic disease, with use in differentiating persistent low levels of hCG from malignant disease.
  • testicular germ cell cancers Three patients suspected of having seminoma or testicular choriocarcinoma, both of which are testicular germ cell cancers, were referred to the USA hCG Reference Service. Samples of urine and serum were obtained from each patient and the level of total hCG was analyzed using DPC Immulite test kit. The samples were then tested for immunoreactivity to ITA using the Nichols Institute Diagnostics: Advantage ITA test. In all three cases, all of the hCG immunoreactivity was due to ITA, showing that these testicular germ cell cancers, like placenta choriocarcinoma, produce ITA.
  • ITA serves as a useful marker to detect both testicular and ovarian germ cell cancers.

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Abstract

L'invention concerne des procédés pour détecter la présence ou l'absence de cellules trophoblastiques invasives. En outre, elle concerne des procédés pour distinguer les maladies trophoblastiques malines ou invasives des maladies latentes, et des procédés pour surveiller l'évolution des maladies trophoblastiques latentes. L'invention concerne des procédés pour détecter la présence ou l'absence des tumeurs de cellules germinales.
PCT/US2003/021306 2002-10-10 2003-07-09 Hcg hyperglycosyle (antigene des trophoblastes invasifs) utilise dans le diagnostic differentiel de la maladie trophoblastique maline ou invasive WO2004033626A2 (fr)

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COLE L.A. ET AL: 'Utility of commonly used commercial human chorionic gonadotropin immunoassays in the diagnosis and management of trophoblastic diseases' CLINICAL CHEMISTRY vol. 47, no. 2, 2001, pages 308 - 315, XP002956462 *
ELLIOT M.M. ET AL: 'Carbohydrate and peptide structure of the alpha- and beta-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma' ENDOCRINE vol. 7, no. 1, August 1997, pages 15 - 32, XP009026237 *

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